Genetic background, nutrition and obesity: a review.

OBJECTIVE: To summarize the latest information on the relationship between genes and common forms of obesity, and to review genetic markers (SNPs and miRNA) that play a role in predisposing to common forms of obesity and related disorders. MATERIALS AND METHODS: We searched PubMed with the following keywords: (obesity[Title/Abstract]) AND predisposition[Title/Abstract]) AND miRNA[Title/Abstract]) OR polymorphism[Title/Abstract]. RESULTS: From the search we obtained a total of 44 gene loci and 48 miRNAs associated with common obesity. CONCLUSIONS: It is now widely accepted that obesity involves interactions between environmental risk factors (physical inactivity, excessive calorie intake, chronic stress, taste perception) and a genetic background of risk. Analysis of the genetic background of obese subjects is therefore an important way to determine the molecular mechanisms controlling the link between food intake and obesity, enabling a better understanding of how these interactions may differ from person to person.

Prevalence of mutations in LEP, LEPR, and MC4R genes in individuals with severe obesity.

Obesity is a major public health concern; despite evidence of high heritability, the genetic causes of obesity remain unclear. In this study, we assessed the presence of mutations in three genes involved in the hypothalamic leptin-melanocortin regulation pathway (leptin, LEP; leptin receptor, LEPR; and melanocortin-4 receptor, MC4R), which is important for energy homeostasis in the body, in a group of patients with severe obesity. For this study, we selected 77 patients who had undergone bariatric surgery and had a pre-operative body mass index (BMI) >35 kg/m2, early onset and a family history of being overweight. Candidate genes were screened by direct sequence analysis to search for rare genetic variations. The common LEP -2548 G/A polymorphism was also evaluated for its influence on the BMI (in obesity patients) and for obesity risk, using a case-control study involving 117 healthy individuals. Two different non-synonymous alterations in MC4R were found in two patients: the p.(Thr112Met), previously described in the literature as a probable gene involved in the obesity phenotype, and the novel p.(Tyr302Asp) variant, predicted to be pathogenic by in silico evaluations and family segregation studies. The LEP -2548 G/A polymorphism was not associated with the BMI or obesity risk. In conclusion, we have reported a novel mutation in MC4R in a family of Italian patients with severe obesity. Screening for MC4R could be important for directing the carriers of mutations towards therapy including partial agonists of the MC4R that could normalize their appetite and inhibit compulsive eating. Next-generation sequencing could be used to clarify the genetic basis of obesity in the future.

PARP activity and NAD concentration in PMC from patients affected by systemic sclerosis and lupus erythematosus.

The enzyme poly(ADP-ribose) polymerase (PARP-1, EC 2.4.2.30) is activated by DNA strand breaks caused by several agents and utilizes NAD to form polyADPR, bound to acceptor proteins. The involvement of PARP-1 in autoimmune diseases has been suggested: antiPARP autoantibodies are described in systemic lupus erythematosus (SLE), DNA strand breaks have been evidenced in systemic sclerosis (SSc). We tested poly(ADP-ribosyl)ation activity and NAD concentration in PMC from patients affected by SLE or SSc and from controls. Lower PARP-1 activity and higher NAD concentration were observed in pathological conditions than controls, supporting the role of PARP-1 activation in modulating NAD concentration.

Are allopurinol and metabolites found in HPRT deficient erythrocytes responsible for increased NAD synthesis?

Aim of this study was to ascertain whether allopurinol, usually administered to hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficient patients, or metabolites abnormally increased in HPRT deficient erythrocytes (NAD, PPribP) could be directly responsible for the reported increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NADsynthetase (NADs) in these patients. No direct effect of the mentioned metabolites was demonstrated.

Metabolic fate of extracellular NAD in human skin fibroblasts.

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.

Enzyme activities leading to NAD synthesis in human lymphocytes.

Pyridine nucleotide levels and the activities of enzymes involved in NAD synthesis (nicotinic acid phosphoribosyltransferase, nicotinic acid- and nicotinamide mononucleotide-adenylyltransferase) have been assayed in human normal lymphocytes by an HPLC method using radioactive or nonradioactive substrates. NAD concentration was 46.4 +/- 17.2 pmol 10(-6) cells, and that of NADP was 14.5 +/- 3.9 pmol 10(-6) cells (mean +/- standard deviation). The adenylyltransferase activity using nicotinic acid mononucleotide as substrate was 1.530 +/- 0.216 nmol h(-1) 10(-6) cells, using nicotinamide mononucleotide was 1.466 +/- 0.354 nmol h(-1) 10(-6) cells. The apparent K(M) values were 0.015 mM for the former substrate and 0.167 mM for the latter. The mean activity of nicotinic acid phosphoribosyltransferase was 0.038 +/- 0.014 nmol h(-1) 10(-6) cells, and the apparent K(M) for nicotinic acid was 0.165 mM. The proposed methods, easy and rapid to perform, are reliable and sensitive, avoiding the use of radiolabels except for NAPRT and displaying a very low activity. The reported findings, together with the previous ones in human erythrocytes, can provide an useful base to investigate NAD metabolism in humans through the study of blood cells.

Hypoxanthine-guanine phosphoribosyltransferase deficiency and erythrocyte synthesis of pyridine coenzymes.

Purine and pyridine metabolism were studied in ten Lesch-Nyhan patients, with virtually no hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in erythrocytes. Increased NAD erythrocyte concentrations were found in all patients. Raised activities of two enzymes catalysing NAD synthesis from nicotinic acid (nicotinic acid phosphoribosyltransferase: NAPRT, and NAD synthetase: NADs) was found in erythrocyte lysates from all patients. The two enzymes had normal apparent Km for their substrates and increased Vmax. The rate of synthesis of pyridine nucleotides from nicotinic acid by intact erythrocytes in vitro was also increased in most patients. These findings suggest that raised NAD concentrations in HPRT- erythrocytes are due to enhanced synthesis as a result of increased enzyme activities.

Thiopurine methyltransferase activity in the erythrocytes of adults and children: and HPLC-linked assay.

A non-radioactive method that uses reverse-phase high performance liquid chromatography is described for the determination of thiopurine methyltransferase (E.C. 2.1.1.67) activity in human erythrocytes. The method is based on the direct quantitation of 6-methyl-mercaptopurine produced from 6-mercaptopurine by crude erythrocyte lysates. The method is accurate and reliable and suitable for diagnostic use. Activity values in control adults ranged from 5 to 32 pmol/h/mg haemoglobin. The activity in the erythrocytes of adult males was significantly higher compared to females (21 +/- 5 and 15 +/- 8 pmol/h/mg haemoglobin, respectively). The activity measured in the erythrocytes of children (22 +/- 5 pmol/h/mg haemoglobin) did not show any significant difference compared to adults. Thiopurine methyltransferase activity was measured in a female patient with systemic sclerosis who developed severe bone marrow depression after treatment with azathioprine and allopurinol. Activity (6.3 +/- 0.5 pmol/h/mg haemoglobin) was found in the lowest range of controls thus supporting the hypothesis that it could be responsible for increased azathioprine cytotoxicity.

Enzymatic activities affecting exogenous nicotinamide adenine dinucleotide in human skin fibroblasts.

The fate of nicotinamide adenine dinucleotide (NAD), AMP, and ADP-ribose supplied to intact human skin fibroblasts was monitored, and the concentrations of intra- and extracellular pyridine and purine compounds were determined by HPLC analysis. Two enzymatic activities affecting extracellular NAD were detected on the plasma membrane, one hydrolyzing the pyrophosphoric bond and yielding nicotinamide mononucleotide (nucleotide pyrophosphatase) and the other cleaving the glycoside link and releasing nicotinamide (NAD-glycohydrolase). No AMP or ADP-ribose was found in the extracellular medium of cells incubated with NAD, the former being completely catabolized to hypoxanthine and the latter degraded to adenine and hypoxanthine.

Purine and pyridine nucleotide metabolism in the erythrocytes of patients with Rett syndrome.

The possible involvement of purine and pyridine metabolism in Rett syndrome, a neurodegenerative disorder of unknown aetiology affecting females, was investigated. The levels of purine and pyridine nucleotides and their metabolites were determined by HPLC in the erythrocytes and plasma of 31 Rett patients and of 17 age-matched controls. Nucleotide production rate from extracellular precursors was determined in intact cells and enzyme activities were assayed in crude lysates using the same HPLC method. Decreased plasma nicotinamide concentrations and lower erythrocyte activities of hypoxanthine phosphoribosyl transferase, adenine phosphoribosyl transferase and phosphoribosylpyrophosphate synthetase were observed in Rett children compared with age-matched controls, while the production rate of IMP from hypoxanthine and of total pyridine nucleotides from nicotinic acid by intact erythrocytes was significantly increased. No significant difference was found in any of the other parameters examined. These findings give a new contribution to the knowledge of the biochemical alterations in Rett syndrome and encourage further investigations in the nucleotide field.

Nicotinic acid phosphoribosyltransferase activity in human erythrocytes: studies using a new HPLC method.

A non-radiochemical method linked to reverse-phase high-performance liquid chromatography was developed to determine the activity of nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) in crude lysates of human red blood cells. The method is accurate and easily reproducible in different chromatographic systems. The enzyme activity was determined in erythrocytes of healthy subjects and in patients with different purine disorders showing altered NAD levels. Very low enzyme activity was found in a boy hemizygous for phosphoribosylpyrophosphate synthetase superactivity, consistent with the low erythrocyte NAD concentration.

An HPLC-linked assay of phosphoribosylpyrophosphate synthetase activity in the erythrocytes of adults and children with neurological disorders.

A two-step non-radioactive method that uses reverse-phase high-performance liquid chromatography (RP-HPLC) is described for the determination of phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) activity in human erythrocytes. The method is accurate and easily reproducible in different chromatographic systems; it is based on the quantification of phosphoribosylpyrophosphate by conversion into orotidine monophosphate and uridine monophosphate. Phosphoribosylpyrophosphate synthetase activity was determined in the erythrocytes of healthy adults and children, the latter showing significantly higher activity than the former. The enzyme activity assayed in children with different neurological disorders was significantly lower in patients with Rett syndrome than in control children or in autistic or mentally retarded patients.

HPLC determination of oxidized and reduced pyridine coenzymes in human erythrocytes.

The nucleotide concentrations in acid and alkaline erythrocyte extracts have been measured by RP-HPLC in healthy controls and in patients bearing different inherited disorders, with altered erythrocyte NAD(P) levels. The objective was the simultaneous determination of the nucleotide profile and of the oxidative state of pyridine coenzymes by the most suitable extraction method. Both alkaline and acid extractions were necessary to obtain the complete pattern, due to defective recovery of the oxidized or reduced coenzymes, respectively, during the extraction procedures. Purine nucleotide quantification seemed to be reliable by all methods. High NADP+ levels were confirmed in two glucose-6-phosphate dehydrogenase deficient patients, coupled with raised NAD levels, lowered NADPH/NADP+ ratio and increased NADH/NAD+ ratio. Higher NAD+ and normal or lower NADH/NAD+ ratios were found in two hypoxanthine-phosphoribosyltransferase deficient patients, while a patient with superactive phosphoribosylpyrophosphate synthetase showed a decreased NADH level in addition to the low NAD+ level previously found.

Nicotinamide mononucleotide adenylyltransferase activity in human erythrocytes.

Nicotinamide mononucleotide adenylyltransferase (NMN-AT) activity has not hitherto been demonstrated in human red blood cells, owing to its low activity. Since it is usually located in the nucleus, the possibility of finding it in human erythrocytes was excluded. Here we report the first demonstration and characterization of NMN-AT in human red blood cells, by an HPLC method. The enzyme is Mg2+ dependent, with a Km of 0.303 mM for nicotinamide mononucleotide and 0.103 mM for ATP, and a Vmax of 346 nmol g Hb-1 h-1. The crude preparation is also active on nicotinic acid mononucleotide, producing nicotinic acid adenine dinucleotide. NMN-AT activity is inhibited by nicotinic acid mononucleotide, and nicotinic acid adenylyltransferase is inhibited by nicotinamide mononucleotide. Fiftyfold purification of NMN-AT was achieved by DEAE-Toyopearl chromatography, and the kinetic characteristics were determined. The partially purified preparation maintained its nicotinic acid adenylyltransferase activity. These findings are discussed in light of the regulation of NAD metabolism in human red blood cells.

[Estrogen receptor levels in the liver of intact and gonadectomized rats and in cultured hepatocytes]. / Livelli recettoriali per gli estrogeni nel fegato di ratti intatti e gonadectomizzati ed in epatociti in coltura.

The concentrations of hepatic estrogen receptor were determined in intact and gonadectomized male and female rats. The hydroxylapatite assay demonstrated that the ablation of gonads induces, in the liver, an increase of estrogen receptors. The data obtained suggests that the synthesis of these receptors in the liver might be estrogen- and androgen-dependent. The same analysis performed on hepatocyte cytosol, derived from ovariectomized females, and cultured for 24, 48 and 72 hours, showed that time of culture is an important factor in determining a decrease of estrogen receptor concentrations in the cells. The results obtained allowed us to conclude that in the maintenance of the normal levels of the hepatic estrogen receptors, either sexual and non-sexual hormones are involved.

Influence of testosterone on purine nucleotide turnover in rat kidney.

The influence of testosterone on purine nucleotide metabolism in rat kidney has been investigated in adult and in prepubertal castrated rats. Results have been evaluated through biomathematical model. Castration enhanced the turnover of purine nucleotides in adult rats and reduced it in young castrated rats. Treatment with testosterone in the castrated rats further enhanced nucleotide turnover both in the adult rats and also in the second group, with an oscillatory profile. A clear effect on the inosinic branch point was demonstrated, and specifically on GMP formation, which was opposite according to the age of the animal. The different behavior in the two groups after castration was partially ascribed to the action of other hormones in the absence of testosterone. The observed changes show that the action of the hormone is not limited to sexual organs; they might be at the basis of variations in cellular size and number which probably occur in the kidney after orchiectomy and following androgen administration.